It is well known that extracellular fluid flow-induced by mechanical loading increases osteoblastic cell metabolism, differentiation, extracellular matrix protein production and gene expression. Recently, there is increasing evidence suggesting that extracellular nucleotides play an important role in bone cell proliferation and differentiation. Thus, the central goal of this project is to characterize the mechanism by which osteoblastic cells detect extracellular fluid flow and transduce this into biochemical responses. Our central hypothesis is that biophysical signals, such as fluid flow, stimulate osteoblastic cell differentiation via a mechanism involving cytosolic calcium mobilization and, importantly, this requires the presence of extracellular nucleotides. We will test this hypothesis using a combination of fluorescent imaging, bioengineering and molecular biological techniques to complete four specific aims. In specific aim 1 we will examine whether extracellular nucleotides are necessary for fluid flow mobilization of cytosolic calcium in osteoblastic cells. In specific aims 2 and 3 we will determine the roles of specific P2Y purinergic receptors in fluid flow-induced increases in intracellular calcium concentration. In the final specific aim we will examine the effects of extracellular nucleotides and their specific receptors (identified in specific aims 1 to 3) on flow-induced osteoblastic differentiation. This will be achieved by quantifying flow-induced effects on three osteoblastic differentiation markers, alkaline phosphatase, osteopontin, and osteocalcin. By completing these specific aims we will gain insights into the mechanism by which biophysical signals affect bone cell behavior. Such information may provide guidance in developing novel treatments for osteopenia